Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions of dollars. Various strategies have been used in attempting to control such pests.
One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. An example of a biopesticide is a bacterium which produces a substance toxic to the infesting pest A biopesticide is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusion(s) may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255).
There are several Bacillus thuringiensis strains that are widely used as biopesticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai have been found to produce delta-endotoxins specific for Lepidoptera. Bacillus thuringiensis subsp. israelensis has been found to produce delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112). Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203), has been found to produce a delta-endotoxin specific for Coleoptera.
Bacillus thuringiensis subsp. tenebrionis has been deposited with the German Collection of Microorganisms under accession number DSM 2803. Bacillus thuringiensis subsp. tenebrionis was isolated in 1982 from a dead pupa of the mealworm Tenebrio molitor (Tenebrionidae, Coleoptera). The strain produces within each cell one spore and one or more pesticidal parasporal crystals which are of flat platelike form with an edge length of about 0.8 .mu.m to 1.5 .mu.m. It belongs to serotype H8a,8b and pathotype C of Bacillus thuringiensis (Krieg et al., 1987, System. Appl. Microbiol. 9, 138-141; Krieg et al., 1988, U.S. Pat. No. 4,766,203). It is only toxic against certain leaf-eating beetle larvae (Chrysomelidae), but ineffective against caterpillars (Lepidoptera), mosquitoes (Diptera) or other insects.
The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Herrnstadt et al., 1986, Bio/Technology 4:305-308; Hermstadt and Soares, 1988, U.S. Pat. No. 4,764,372). This strain, designated "Bacillus thuringiensis subsp. san diego", M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis. Furthermore, the '372 patent has been assigned to Novo Nordisk A/S. A spo-cry.sup.+ (asporogenous crystal forming) mutant of M-7 has purportedly been obtained by culturing M-7 in the presence of ethidium bromide (Hermstadt and Gaertner, 1987, EP Application No. 228,228). However, there was no indication of increased production of delta-endotoxin, increased parasporal crystal size, and/or increased pesticidal activity relative to the parental, M-7 strain.
The crystal proteins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The six major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), Diptera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255), Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form. Various cry genes have been cloned, sequenced, and expressed in various hosts, e.g., E. coli (Schnepf et al., 1987, J. Bacteriol. 169:4110-4118), Bacillus subtilis (Shivakumar et al., 1986, J. Bacteriol. 166:194-204), and maize plants (Koziel et al., 1993, Bio/Technology 11:194-200).
Amplification of cry genes has been achieved in Bacillus subtilis. The delta-endotoxin gene of Bacillus thuringiensis subsp. kurstaki HD73 has been cloned into Bacillus subtilis using an integrative plasmid and amplified (Calogero et al., 1989, Appl. Environ. Microbiol. 55:446-453). However, no increase in crystal size was observed as compared to Bacillus thuringiensis subsp. kurstaki HD73. Furthermore, no difference in pesticidal activity was reported.
The level of expression of delta-endotoxin genes appears to be dependent on the host cell used (Skivakumar et al., 1989, Gene 79:21-31). For example, Skivakumar et al. found significant differences in the expression of the cryIA and cryIIA delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium. The cryIA gene was expressed when present on a multicopy vector in Bacillus megaterium, but not in Bacillus subtilis. The cryIIA gene was expressed in both hosts, but at a higher level in Bacillus megaterium. Sections of Bacillus megaterium cells expressing these delta-endotoxin genes were examined by electron microscopy; the presence of large bipyramidal crystals in these cells was detected. However, there is no indication that these crystals are any larger than crystals found in Bacillus thuringiensis subsp. kurstaki which normally contain these genes. Results from bioassays of the Bacillus megaterium cells expressing these delta-endotoxin genes indicate that there was no increase in pesticidal activity as compared to Bacillus thuringiensis subsp. kurstaki. Indeed, five times the concentration of Bacillus megaterium than Bacillus thuringiensis subsp. kurstaki was required to obtain the same insect killing effect.
Recombinant Bacillus thuringiensis strains have also been disclosed. Shuttle vectors with various copy numbers containing the cryIIIA gene, which encodes a delta-endotoxin protein specific for pests of the order Coleoptera, were constructed and transformed into Bacillus thuringiensis subsp. kurstaki HD1 Cry-B (Arantes and Lereclus, 1991, Gene 108:115-119). It was found that when the gene expression level and vector copy number were compared, a plateau in delta-endotoxin production was reached with a copy number of about fifteen per equivalent chromosome. The crystal size and pesticidal activity of these recombinants were not determined or disclosed in that reference.
Lecadet et al. (1992, Appl. Environ. Microbiol 58:840-849) and Lereclus et al. (1992, Bio/Technology 10:418-421) disclose the construction of various recombinant Bacillus thuringiensis strains expressing the cryIA(a) and/or the cryIIIA genes. Those strains with dual specificities possessed pesticidal activity corresponding to those of the parental strains. In one instance, the cryIIIA gene was introduced via transduction into a heterologous cry-strain. In this instance, the pesticidal activity was increased relative to Bacillus thuringiensis subsp. tenebrionis; a larger crystal was also observed (Lecadet et al., 1992, Appl. Environ. Microbiol. 58:840-849). Lecadet et al. attributed the hyperexpression of the CryIIIA protein to the release of the cryIIIA gene from negative regulation in the heterologous strain.
The utility of Bacillus thuringiensis strains for the control of pests of the orders Lepidoptera, Diptera, and Coleoptera is dependent upon efficient and economical production of the crystal delta-endotoxin(s) and the potency of the product produced. This, in turn, is dependent upon the amount of crystal delta-endotoxin(s) which can be produced by fermentation of the Bacillus thuringiensis strains.
Bacillus thuringiensis has been used for many years for the production of pesticides. Mutants of Bacillus thuringiensis have also been disclosed. Generally, such mutants have been obtained using classical mutagenesis. There has been disclosed, for example, a mutant of Bacillus thuringiensis subsp. tenebrionis which produces a crystal delta-endotoxin with a larger crystal size and greater pesticidal activity as compared to a corresponding parental strain (Gurtler and Petersen, 1994, U.S. Pat. No. 5,279,962).
Mutants producing crystal delta-endotoxins with a larger crystal size and increased pesticidal activity would give a more efficient and economical production of Bacillus thuringiensis crystal delta-endotoxin(s), and a possibility for manufacture of Bacillus thuringiensis products with increased potency at equal or lower cost. This, in turn, would be an advantage for the user as reduced volumes of pesticide formulation have to be stored and handled for a given acreage. In addition, the users will have less container material to dispose of, thereby, reducing the impact on the environment.
For example, in controlling beetle larvae, Bacillus thuringiensis subsp. tenebrionis crystal delta-endotoxin preparations have been of relatively low potency or strength requiring the application of relatively large amounts of the preparations to areas to be treated, such as 5 to 10 liter/ha compared to 1 to 2 liter/ha of most other Bacillus thuringiensis products and most other insecticides. It is advantageous to obtain products with increased pesticidal activity. Consequently, a recognized need for products of improved strength exists.
One way to fulfill this need is to concentrate the preparations. However, concentration adds considerably to the production cost in comparison to the savings obtained in storage and transportation. And, in some cases, concentration to obtain a pesticidally acceptable level is not achievable or practical.
A more expedient solution would be to create integrants of existing Bacillus thuringiensis strains which produce substantially larger quantities of crystal delta-endotoxin with greater pesticidal activity compared to wild-type strains.
The art has strived to improve the effectiveness and to broaden the host range of Bacillus thuringiensis. Means have included isolating Bacillus thuringiensis strains with new or improved toxicity, engineering present Bacillus thuringiensis strains, and designing more effective formulations by combining Bacillus thuringiensis crystal delta-endotoxins and spores with new pesticidal carriers or with chemical pesticides.